Identification and characterization of the key lethal toxin from jellyfish Cyanea nozakii.

Jellyfish Cyanea nozakii venom is a complex mixture of various toxins, most of which are proteinous biological macromolecules and are considered to be responsible for clinical symptoms or even death after a severe sting. Previous transcriptome and proteome analysis identified hundreds of toxins in the venom, including hemolysins, C-type lectin, phospholipase A2, potassium channel inhibitor, metalloprotease, etc. However, it is not clear which toxin in the venom plays the most important role in lethality. Herein, we isolated the key lethal toxin (Letoxcn) from jellyfish Cyanea nozakii using anion exchange chromatography, size-exclusion chromatography, and cation exchange chromatography. The molecular weight of Letoxcn is ∼50 kDa with the N-terminal sequences of QADAEKVNLPVGVCV. Peptide mass fingerprinting analysis of Letoxcn shows that it may have some motifs of phospholipase, metalloproteinase, thrombin-like enzyme, potassium channel toxin, etc. However, only metalloproteinase activity but no hemolytic, PLA2, or blood coagulation activity was observed from in vitro toxicity analysis. Overall, this study uncovered and characterized the key lethal toxin in the venom of jellyfish Cyanea nozakii, which will not only help to reveal the molecule mechanism of the lethality, but also develop effective treatment like antivenom for this jellyfish sting in the future.

Preparation and Neutralization Efficacy of Novel Jellyfish Antivenoms against Cyanea nozakii Toxins.

Jellyfish stings are a common issue globally, particularly in coastal areas in the summer. Victims can suffer pain, itching, swelling, shock, and even death. Usually, hot water, vinegar, or alumen is used to treat the normal symptoms of a jellyfish sting. However, a specific antivenom may be an effective treatment to deal with severe jellyfish stings. Cyanea nozakii often reach a diameter of 60 cm and are responsible for hundreds of thousands of stings per year in coastal Chinese waters. However, there has been no specific C. nozakii antivenom until now, and so the development of this antivenom is very important. Herein, we collected C. nozakii antisera from tentacle extract venom immunized rabbits and purified the immunoglobulin (IgG) fraction antivenom (AntiCnTXs). Subsequently, two complete procedures to produce a refined F(ab')2 type of antivenom (F(ab')2-AntiCnTXs) and Fab type of antivenom (Fab-AntiCnTXs) by multiple optimizations and purification were established. The neutralization efficacy of these three types of antivenoms was compared and analyzed in vitro and in vivo, and the results showed that all types of antibodies displayed some neutralization effect on the lethality of C. nozakii venom toxins, with the neutralization efficacy as follows: F(ab')2-AntiCnTXs ≥ AntiCnTXs > Fab-AntiCnTXs. This study describes the preparation of novel C. nozakii jellyfish antivenom preparations towards the goal of developing a new, effective treatment for jellyfish stings.

Evaluation of the complete nuclear rDNA unit sequence of the jellyfish Cyanea nozakii Kishinouye (Scyphozoa: Semaeostomeae) for molecular discrimination.

The harmful jellyfish Cyanea nozakii Kishinouye has frequently occurred on Korean coasts, and its blooms have caused serious ecological and economic damages. DNA sequences of the C. nozakii for molecular detection and discrimination are relatively scarce. In this study, we determined the complete sequence of a single unit of tandemly repeated ribosomal DNA (rDNA) of the Korean C. nozakii and characterized the molecular features of the rDNA. The complete rDNA contained 8,003 bp (48.4% GC) with the same gene arrangement (18S, ITS1, 5.8S, ITS2, 28S, and IGS) to the typical eukaryotes. Dot plot analysis showed that the coding regions (18S, 5.8S, and 28S) were highly conserved, while the non-coding regions (ITS1, ITS2, and IGS) were more variable and parsimony-informative. The IGS contained a putative transcription termination signal (poly(T) tract) and four repeats of block minisatellites. Phylogenetic analyses using 18S and 28S rDNA revealed well-resolved relationships of C. nozakii within the order Semaeostomeae, separating it from other Cyanea species. The complete rDNA sequence provides various options for the selection of jellyfish taxonomic markers and may be useful for discriminating between species of C. nozakii and phylogeny reconstruction with close relatives.

Updated descriptions of the nematocysts of the scyphozoan jellyfish Cyanea nozakii Kishinouye, 1891 (Cnidaria, Scyphozoa).

Nematocysts are typical cnidarian organelles that can discharge and release venom under physicochemical stimuli for predation and defense. Cyanea nozakii Kishinouye, 1891, a dominant jellyfish-blooming species in Chinese coastal waters, possesses numerous stinging nematocysts on its tentacles, and causes many envenomations every year. However, detailed taxonomic information of nematocysts in C. nozakii is elusive. In the present study, the morphological characteristics of nematocysts from C. nozakii were examined by combined light and scanning-electron microscopy. Five nematocyst types were revealed in the cnidome of C. nozakii, including common nematocyst types identified in Scyphozoa: atrichous isorhizas (a-atrich/O-atrich), microbasic euryteles, and microbasic birhopaloids type II. Importantly, two seldom reported types, microbasic b-mastigophores and microbasic birhopaloids type I, were also found, for the first time, in the cnidome of C. nozakii. This study contributes to understanding of the cnidome of C. nozakii, and the present study strongly suggests that the nematocysts of Scyphozoa are more diverse and complex than previously reported, which sheds new light on the nematocyst types in Scyphozoa species.

Combined Proteome and Toxicology Approach Reveals the Lethality of Venom Toxins from Jellyfish Cyanea nozakii.

Jellyfish are a type of poisonous cnidarian invertebrate that secrete lethal venom for predation or defense. Human beings often become victims of jellyfish stings accidentally while swimming or fishing and suffer severe pain, itching, swelling, inflammation, shock, and even death. Jellyfish venom is composed of various toxins, and the lethal toxin is the most toxic and hazardous component of the venom, which is responsible for deaths caused by jellyfish stings and envenomation. Our previous study revealed many toxins in jellyfish venom, including phospholipase A2, metalloproteinase, and protease inhibitors. However, it is still unknown which type of toxin is lethal and how it works. Herein a combined toxicology analysis, proteome strategy, and purification approach was employed to investigate the lethality of the venom of the jellyfish Cyanea nozakii. Toxicity analysis revealed that cardiotoxicity including acute myocardial infarction and a significant decrease in both heart rate and blood pressure is the primary cause of death. Purified lethal toxin containing a fraction of jellyfish venom was subsequently subjected to proteome analysis and bioinformation analysis. A total of 316 and 374 homologous proteins were identified, including phospholipase A2-like toxins and metalloprotease-like toxins. Furthermore, we confirmed that the lethality of the jellyfish venom is related to metalloproteinase activity but without any phospholipase A2 activity or hemolytic activity. Altogether, this study not only provides a comprehensive understanding of the lethal mechanism of jellyfish venom but also provides very useful information for the therapeutic or rescue strategy for severe jellyfish stings.

The complete mitogenome of ghost jellyfish Cyanea nozakii (Cnidaria, Semaeostomeae, Cyaneidae).

The cnidarian jellyfishes are impressive organisms to show animal mitochondrial genomic diversities. Their mitogenome structure is linear and tRNA content has one or two in numbers, which is highly different than other metazoans. In this study, a complete mitogenome of the ghost jellyfish Cyanea nozakii (Cnidaria, Semaeostomeae, Cyaneidae) was sequenced and analyzed. The mitgenome is 17,381 bp long with 38.5% A, 16.0% C, 13.9% G, and 31.6% T nucleotide distributions. In addition, phylogenetic relationship of C. nozakii in the class Scyphozoa was investigated by using mitochondrial protein coding genes. Due to results, C. nozakii was positioned in the paraphyletic order Semaeostomeae. This is the first complete mitogenome from the genus Cyanea.

Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms' Lytic Activity Using Mass Spectrometry and Zymography.

BACKGROUND: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. METHODS: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. RESULTS: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14-18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. CONCLUSION: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

Biochemical and kinetic evaluation of the enzymatic toxins from two stinging scyphozoans Nemopilema nomurai and Cyanea nozakii.

Jellyfish envenomations are emerging as an important public health concern occurred worldwide. In China, the situation is getting worse with numerous people stung by jellyfish Nemopilema nomurai (N. nomurai) and Cyanea nozakii (C. nozakii) in the summer. However, the proteinaceous mixtures in nematocysts responsible for the symptoms of jellyfish stings were scarcely characterized and understood in view of enzymatic constituents and toxicity. In the present study, enzymatic properties of jellyfish N. nomurai and C. nozakii nematocyst venom were analyzed biochemically and kinetically. The current data revealed that N. nomurai and C. nozakii nematocyst venom exhibited various enzymatic activities, of which metalloproteinases activity and PLA2s-like activity were predominant. Moreover, the catalytic activities of metalloproteinases and PLA2s-like were dependent on different physiochemical conditions such as temperature, pH and divalent ions. Kinetic profiling revealed their catalytic behaviors fitted the Michaelis-Menten equation under specific conditions. Findings suggested jellyfish nematocyst venom possessed diverse enzymatic constituents, which may underlie the extensively characterized bioactivities of jellyfish venom and human envenomations. Hence, our study will contribute to understanding the enzymatic constituents and toxicity of jellyfish nematocyst venom and may afford potential therapeutic targets for developing drugs for jellyfish stings.

Combined proteomics and transcriptomics identifies sting-related toxins of jellyfish Cyanea nozakii.

UNLABELLED: Jellyfish sting has become a worldwide issue of critical concern to human health and safety in coastal areas in recent decades. Cyanea nozakii is one of the dominant blooming species and dangerous stingers in China. However, it remains unclear how many and what types of toxins are present in the venom. So, we used a combined transcriptomics and proteomics approach to investigate the venom composition of jellyfish C. nozakii. In total 4,608,524 Illumina valid reads were obtained to de novo assemble to 40,434 unigenes in the transcriptomics analysis. And, a total of 311,635 MS/MS spectra with 12,247 unique MS/MS spectra were generated to 1556 homologous proteins in the proteomics analysis. 174 potential toxin proteins were identified, with 27 proteins homology to the toxins from venomous animals, including phospholipase A2, zinc metalloproteinase-disintegrin agkistin, serine protease inhibitor, plancitoxin-1, alpha-latrocrustotoxin-Lt1a, etc. This study described the transcriptomics and venom proteomics of jellyfish C. nozakii for the first time. Our findings provide a comprehensive understanding of the venom composition of C. nozakii. Furthermore, the results may also be very helpful for the discovery of novel bioactive proteins, as well as the development of effective treatments for jellyfish sting in the future. BIOLOGICAL SIGNIFICANCE: Jellyfish Cyanea nozakii is one of the most dangerous stingers in the coast of china. Hundreds of thousands of people would be stung every year and victims suffered a severe pain, itch, swelling, inflammation, wheal and even more serious consequence. However, it remains unclear how many and what types of toxins are present as well as the relationship between the clinical symptoms and toxins. Our combined transcriptomics and proteomics findings can provide a comprehensive understanding of the venom composition of C. nozakii and will also be helpful for the development of effective treatments for jellyfish sting in the future.

Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method.

Blooms of the harmful jellyfish Cyanea nozakii, which are a severe nuisance to fisheries and tourisms, frequently occur in the northern East China Sea, Yellow Sea, and Bohai Sea. To provide early warning of this species, a simple and effective molecular method for identifying C. nozakii was developed using the loop-mediated isothermal amplification method (LAMP). The LAMP assay is highly specific and uses a set of four primers that target six different regions on the mitochondrial cytochrome c oxidase subunit I (COI) gene of C. nozakii. The amplification conditions, including the dNTP and betaine concentrations, the inner primer to outer primer concentration ratio, reaction time and temperature, were optimized. The LAMP assay amplified DNA extracted from tissue samples of C. nozakii but did not amplify DNA from other common scyphozoans and hydrozoans collected in the same region. In addition, the LAMP assay was more sensitive than conventional PCR. Therefore, the established LAMP assay is a sensitive, specific, fast, and easily performed method for detection of C. nozakii at different stages in their life cycle.

Jellyfish (Cyanea nozakii) decomposition and its potential influence on marine environments studied via simulation experiments.

A growing body of evidence suggests that the jellyfish population in Chinese seas is increasing, and decomposition of jellyfish strongly influences the marine ecosystem. This study investigated the change in water quality during Cyanea nozakii decomposition using simulation experiments. The results demonstrated that the amount of dissolved nutrients released by jellyfish was greater than the amount of particulate nutrients. NH4(+) was predominant in the dissolved matter, whereas the particulate matter was dominated by organic nitrogen and inorganic phosphorus. The high N/P ratios demonstrated that jellyfish decomposition may result in high nitrogen loads. The inorganic nutrients released by C. nozakii decomposition were important for primary production. Jellyfish decomposition caused decreases in the pH and oxygen consumption associated with acidification and hypoxia or anoxia; however, sediments partially mitigated the changes in the pH and oxygen. These results imply that jellyfish decomposition can result in potentially detrimental effects on marine environments.

Characterisation of acid-soluble and pepsin-solubilised collagen from jellyfish (Cyanea nozakii Kishinouye).

Annual outbreaks of the Jellyfish (Cyanea nozakii Kishinouye) in the waters of the Yellow Sea and the East China Sea are regarded as a nuisance. Thus, utilizing this jellyfish species is of great significance to reduce harm to fisheries and marine environments. The yield of the acid-soluble collagens (ASCs) from the C. nozakii umbrella was 13.0% (dry weight) and that of the pepsin-solubilised collagens (PSCs) was 5.5% (dry weight). The SDS-PAGE patterns of the ASCs and PSCs differed from that of type I collagen, which indicate the presence of (α1)3. The denaturation temperature (Td) of the collagens was approximately 23.8°C. Fourier transform infrared spectroscopy proved that the ASCs and PSCs retained their helical structures and the As, Pb, and Hg content of the collagens, detected by ICP-MS, were considerably lower than the national standards. The results suggest that collagens isolated from C. nozakii can potentially be used as an alternative source of collagen for use in various applications.